1. Field of the Invention
This invention relates to apatite-based chromatographic resins and their use in the purification of proteins and other target molecules from biological samples.
2. Description of the Prior Art
Apatite in its various forms, examples of which are hydroxyapatite, ceramic hydroxyapatite, fluoroapatite, and fluoride-enhanced apatite, is used as a chromatographic solid phase in the separation and purification of a wide variety of target molecules by way of binding mechanisms that involve either affinity, ion exchange or hydrophobic interactions, or combinations of these mechanisms. Apatite-based chromatography resins are particularly useful in protein purifications, notably purifications of recombinant proteins from host cell proteins, aggregates, endotoxin, and DNA. Sample loading of an apatite-based resin, particularly with proteins, is most often conducted by first equilibrating the resin to pH 6.5 with phosphate buffer at 2 mM to 5 mM, and then loading the sample in a solution at the same pH and buffer content. Once the sample is loaded, unbound components are washed from the resin and the target molecule is eluted with an elution buffer that typically contains an alkali metal salt, most often at pH of 8.0 or below. The equilibration and loading buffers both saturate the hydroxyapatite surface with hydroxonium ions (H3O+), which are then desorbed upon exposure to the elution buffer. This desorption causes the resin to deteriorate over time, resulting in a loss of resin mass and a decline in the particle strength of the resin. Of potential relevance to this issue is the disclosure of commonly owned, co-pending U.S. patent application Ser. No. 13/205,354, filed Aug. 8, 2011, entitled “Elution of Proteins From Hydroxyapatite Resins Without Resin Deterioration,” inventor L. J. Cummings. That disclosure describes the use of elution buffers that include calcium ions and phosphate ions at a pH of 6.0 or below, thereby both eluting the protein and treating the resin with these ions at the same time. While this buffer is effective in inhibiting the resin deterioration that occurs without the inclusion of the calcium ions in the buffer, the exposure of the resin to the calcium ions in this method is limited to the duration of the elution step and the volume of the elution buffer, and the product eluate, although lacking the impurities in the original sample, contains calcium ions.